The growth and differentiation of cells depends on cytoplasmic proteins that carry signals from activated growth factor receptors. These signaling proteins are modular in architecture, consisting of individual structural domains that mediate molecular recognition events. By elucidating the atomic resolution structure of new signaling domains, the principles that govern cell growth and differentiation can be understood and predicted. In this proposal we use multidimensional heteronuclear magnetic resonance spectroscopy to characterize the structure and interactions of a new signaling module, the Eps15 homology (EH) domain. The EH domain contains 95 amino acids and mediates interactions between signaling proteins. In the past three years, fourteen proteins containing 28 EH domains have been found in organisms ranging from yeast to man. EHG domain-containing proteins are involved in growth factor receptor sorting and internalization, dephosphorylation, and interactions with src homology 3 domains. Eps15 (epidermal growth factor receptor pathway substrate clone 15) is the prototypic member of the EH family of proteins and its biochemical interactions and transforming activity have been established. Fragments of human Eps15 have been expressed and purified in order to pursue the following aims: 1: Determine the oligomeric state of the EH domain. Eps15 and other EH domain-containing proteins assemble into multimeric protein complexes. Sedimentation equilibrium, dynamic light scattering and chemical crosslinking experiments have been performed to characterize the oligomeric states of Eps15 fragments. 2. Elucidate the three dimensional structure of the EH domain. Due to the absence of significant similarity to known protein structures it is likely that the EH domain has a novel fold. Analysis of heteronuclear NMR spectra of an EH domain monomer has already revealed several alpha helices and beta strands as well as sequential assignments. 3: Characterize the structural relationship between tandem EH domains. Most EH domain- containing proteins contain two or three tandem EH repeats that may interact cooperatively with each other. To establish how adjacent EH domains influence each other, the structures of dual and triple EH domain constructs will be determined. 4: Investigate the protein binding mechanism of the EH domain. EH domains mediate protein interactions that are thought to be regulated by calcium and tyrosine phosphrylation. Surface plasmon resonance detection and co- precipitation assays will be used to identify ligands of the EH domain. Peptide binding interactions of unmodified and phosphorylated forms of EH domains will be structurally characterized in the presence and absence of calcium.